The development of safe an effective topical microbicides for vaginal and rectal use could play a critical role in reducing human immunodeficiency virus (HIV) transmission rates worldwide. PRO 2000, a naphthalene sulfonic acid, is active against a wide range of HIV isolates in vitro, and also inhibits herpes simplex virus (HSV), a major cofactor in HIV transmission. A combination of PRO 2000 with a second microbicide that targets a different step in the HIV life cycle might have distinct advantages over either drug alone, including enhanced efficacy, reduced mucosal inflammation, and possibly, an expanded spectrum of activity against HIV and other sexually transmitted pathogens. The focus of this Program, therefore, is to identify optimal combinations that act additively or synergistically with PRO 2000 and provide even greater protection. A valuable lesson learned from the nonoxynol-9 (N-9) trials is that the use of topical microbicides may alter innate immune responses of the female genital tract at a mucosal level and enhance sexual transmission of HIV. It is clear that early pilot in vivo studies are needed prior to the initiation of large-scale clinical trials. Although monitoring for cervicovaginal lesions has been a routine part of clinical safety microbicide trials, little is known about potential subtle changes in the cervicovaginal mucosal barrier, including induction of mucosal inflammation. In Project 3, three pilot human clinical studies will be conducted to examine the effects of PRO 2000 on mucosal immunity and on genital tract HIV. In Specific Aim 1, the effects of PRO 2000 on mediators of inflammation will be evaluated. Healthy participants will apply PRO 2000 or placebo gel intravaginally daily for fourteen days and cell populations and cytokines associated with inflammation will be measured. Microbicides may be used by women knowingly or unknowingly infected with HIV. Therefore, a pilot clinical study will be conducted in Specific Aim 2 to assess the effect of PRO 2000 on infectious HIV-1 recovered from cervicovaginal lavage fluid. The impact of repeated application of PRO 2000 on HIV-infected women will be assessed in Specific Aim 3. HIV-infected women will apply PRO 2000 gel or a matched placebo daily for fourteen consecutive days and cell populations and cytokines associated with vaginal inflammation will be measured. The effect of PRO 2000 on genital tract viral RNA and DNA will also be determined. The impact on cell-associated HIV nucleic acid will be assessed using a laboratory-developed PCR-based assay for circular viral DNA, a surrogate marker for local de novo replication. This work will lay the groundwork for future clinical trials with combination microbicides identified in Projects 1 and 2.